Vol. 133, No. 3

JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1096-1107 0021-9193/78/0133-1096$02.00/0 Copyright ) 1978 American Society for Microbiology

Printed in U.S.A.

Arginine Metabolism in Saccharomyces cerevisiae: Subcellular Localization of the Enzymes JEAN-CLAUDE JAUNIAUX,* L. ANTONIO URRESTARAZU, AND JEAN-MARIE WLAME Laboratoire de Micro biologie, Faculte des Sciences, Universite Libre de BruxeUes, and Institut de Recherches du Centre d'Enseignement et de Recherches des Industries Alimentaires et Chimiques, B-1070 Brussels, Belgium Received for publication 19 September 1977

Subcellular localization of enzymes of arginine metabolism in Saccharomyces cerevisiae was studied by partial fractionation and stepwise homogenization of spheroplast lysates. These enzymes could clearly be divided into two groups. The first group comprised the five enzymes of the acetylated compound cycle, i.e.,

acetylglutamate synthase, acetylglutamate kinase, acetylglutamyl-phosphate reductase, acetylornithine aminotransferase, and acetylornithine-glutamate acetyltransferase. These enzymes were exclusively particulate. Comparison with citrate synthase and cytochrome oxidase, and results from isopycnic gradient analysis, suggested that these enzymes were associated with the mitochondria. By contrast, enzymatic activities going from ornithine to arginine, i.e., arginine pathwayspecific carbamoylphosphate synthetase, ornithine carbamoyltransferase, argininosuccinate synthetase, and argininosuccinate lyase, and the two first catabolic enzymes, arginase and ornithine aminotransferase, were in the "soluble" fraction of the cell. In Saccharomyces cerevisiae, and probably in tion by arginase binding. Obligate aerobes, such all Saccharomyces species and a few other yeast as Debaryomyces hansenii, are devoid of such genera, ornithine carbamoyltransferase and car- regulation. Preferential fermenters, such as S. bamoylphosphate synthetase are extramito- cerevisiae, are almost all endowed with this chondrial enzymes (45). In liver (6, 14), in Neu- regulatory system. In the intermediary group rospora (48), and in other yeasts (45) they are which respire preferentially, such as Hansenula mitochondrial. The meaning of this difference in anomala, the regulatory system may or may not be present (50). It has been shown that in yeasts localization is not obvious. The origin of our interest in ornithine carba- such as Schizosaccharomyces pombe, Hansenmoyltransferase localization arises from the oc- ula anomala, and Debaryomyces hansenii, currence in certain yeasts, among them S. cere- where the control by arginase binding is absent, visiae, of a regulatory mechanism termed "epiar- ornithine carbamoyltransferase and carbamoylginasic regulation" (26). This regulation is phosphate synthetase are mitochondrial (45), a achieved through the stoichiometric and revers- situation identical to that of Neurospora (48) ible binding of arginase to ornithine carbamoyl- and mammalian liver cells (14). So far, in yeasts, transferase when both effectors, arginine and the two different localization patterns of the ornithine, are present. This binding leads to a enzymes concerned are correlated with the complete inhibition of ornithine carbamoyl- epiarginasic regulation and, less tightly, with the transferase, whereas arginase activity remains mode of energy production. The type of energy unchanged, so that arginine biosynthesis is production is very likely the main variable in turned off when arginine catabolism is operating these phenomena. With the hope of better defin(25, 29, 30). This regulatory system implies a ing the influence of energy metabolism upon common cellular localization ofthe two enzymes. enzyme location, we undertook to determine Indeed, at variance with the situation existing in whether the localization shift also affects other all other organisms studied, the ornithine car- enzymes of arginine metabolism in S. cerevisiae bamoyltransferase and the arginine pathway- as compared with Neurospora. In S. cerevisiae (Fig. 1) arginine biosynthesis specific carbamoylphosphate synthetase of S. cerevisiae are extramitochondrial enzymes, as is starts from glutamate and acetylcoenzyme A (CoA) and first produces ornithine through the arginase (45). Besides, there is a relation between the energy five acetylated steps, the acetyl group being status of yeasts and the existence of the regula- recycled. Ornithine reacts with carbamoylphos1096

VOL. 133, 1978


FIG. 1. Spatial organization of arginine metabolism and some related reactions in the cell of S. cerevisiae. Continuous thin arrows represent anabolic enzymatic reactions, continuous heavy arrows represent catabolic enzymatic conversions, and dashed arrows represent metabolic translocations. Numbers denote enzymes: I, acetylglutamate synthase (acetyl coenzyme A:L-glutamate N-acetyltransferase, EC; II, acetylglutamate kinase (ATP:N-acetyl-L-glutamate 5-phosphotransferase, EC; III, acetylglutamyl-phosphate reductase (N-acetyl-L-jlutamate-5-semialdehyde:NADP+ oxidoreductase [phosphorylating], EC; IV, ac-

etylornithine aminotransferase (N"-acetyl-L-ornithine:2-oxoglutarate aminotransferase, EC; V, acetylornithine-glutamate acetyltransferase (N"acetyl-L-ornithine:L-glutamate N-acetyltransferase, EC; VI, carbamoylphosphate synthetase (ATP:carbamate phosphotransferase [dephosphorylating, amido transferring], EC; VII, ornithine carbamoyltransferase (carbamoylphosphate:Lornithine carbamoyltransferase, EC; VIII, argininosuccinate synthetase (L-citrulline; L-aspartate ligase, AMP forming, EC; IX, argininosuccinate lyase (L-argininosuccinate arginine lyase, EC 4.32.1); X, arginase (L-arginine amidinohydrolase, EC; XI, ornithine aminotransferase (L-ornithine:2-oxo-acid aminotransferase, EC; XII, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 7); XIII, pyrroline dehydrogenase (1-pyrrooxidoreductase, EC line-5-carboxylate:NAD+; XIV, glutamate dehydrogenase NAD+ (Lglutamate:NAD+ oxidoreductase [deaminating], EC; XV, glutamate dehydrogenase NADP+ (Lglutamate:NADP+ oxidoreductase [deaminating], EC; XVI, L-glutamate kinase; XVII, L-glutamyl-phosphate reductase; XVIII, pyrroline-5-carboxylate reductase (L-proline:NAD[PJ+ 5-oxidoreductase, EC; XIX, proline oxidase. Abbreviations: acCoA, acetyl coenzyme A; acGL U, acetylglutamate; acGLU-P, acetylglutamyl-phosphate; acGLU-SA, acetylglutamic-y-semialdehyde; acORN, acetylornithine; GLU, glutamate; ORN, ornithine; CAP, carbamoylphosphate; CIT, citrumine; ASA, argininosuc-


phate in the ornithine carbamoyltransferase step and gives citrulline. Citrulline is transformed into argininosuccinic acid by argininosuccinic acid synthetase, and argininosuccinate is cleaved by a lyase, giving arginine. In Neurospora acetylglutamate synthase has not yet been studied. The other reactions needed for citrulline production, including the synthesis of carbamoylphosphate, are mitochondrial except for acetylglutamate kinase, which seems to be cytoplasmic (7). Steps between citrulline and arginine as well as arginase, ornithine aminotransferase, and ornithine decarboxylase reactions are in the soluble fraction (48). If in Saccharomyces, in addition to ornithine carbamoyltransferase and carbamoylphosphate synthetase, acetylglutamate kinase is also cytosolic, one may expect a cytosolic localization of the other enzymes of the acetylated compound cycle as well. Thus, new variations in the localization of some arginine enzymes can be expected between Neurospora and Saccharomyces. On the other hand, arginine metabolism in Saccharomyces shows a spatial complexity due to the vacuolar sequestration of arginine, ornithine, and citrulline (51). The subcellular localization of the arginine pathway enzymes will also determine the cellular compartment where the metabolites concerned are produced and which intracellular permeation barriers they have to cross. In vitro studies have shown that the acetylglutamate synthase, a mitochondrial enzyme (52), and the acetylglutamate kinase (9, 17) are inhibited by arginine. A proof of the physiological meaning of this inhibition has not yet been given, but the results strongly suggest a mechanism of feedback inhibition. This raises the problem of the penetration of arginine into mitochondria. Thus, the localizations of acetylglutamate kinase, acetylglutamyl-phosphate reductase, acetylornithine aminotransferase, acetylornithineglutamate acetyltransferase, argininosuccinate synthetase, argininosuccinate lyase, and ornithine aminotransferase were studied. Although the localizations of arginase, ornithine carbamoyltransferase, arginine pathway-specific carbamoylphosphate synthetase (45), and acetylglutamate synthase (52) are already known, they are included in this work for unity of presentation. cinate; ARG, arginine; PUT, putrescine; GLU-SA,

glutamic--y-semialdehyde; PCA, 1-pyrroline-5-carboxylate; PRO, proline; GL U-P, glutamyl-phosphate; KGLU, a-ketoglutarate; AM, ammonia. Symbols: +, enzymes not yet determined; *, the NAD+ and NADP+ glutamate dehydrogenases have been shown to be cytosolic (18, 31).




MATERIALS AND METHODS Yeast strains. All strains of S. cerevisiae were derived from the usual wild type of this laboratory, X1278b (a). The mutant MG790 lacks N-acetylglutamate kinase activity (17). Mutant 100c7, an argininosuccinate synthetase-less mutant, is from our collection. Mutant 7340c, used in enzyme localization studies, bears an isoleucine-valine leaky mutation and the regulatory argRII-51 mutation conferring maximum derepressed levels for the enzymes involved in arginine biosynthesis (2, 11). Bacterial strain. Strain P4XB2A42, a derepressed (argR) mutant of Escherichia coli lacking acetylornithinase activity, was a gift of N. Glansdorff. Cultures and media. Cells were grown at 29°C under conditions described earlier (32), in 3 to 6 liters of minimal medium no. 170 supplemented with vitamins (43), traces of metals (43), sodium pyruvate (pH 3.3) at a final concentration of 0.5%, (NH4)2S04 (20 mM), and Tween 80 at a final concentration of 0.1% to avoid clumps. All solutions were sterilized separately before mixing. Medium 170 is identical in composition to medium 149 (43) except that it did not contain traces of metals and that it was buffered with citric acid-KOH at pH 3.3 instead of the usual pH 6.2. For carbamoylphosphate synthetase studies, uracil (25 ,ug/ml) was added to our usual medium to repress the pyrimidine pathway-specific carbamoylphosphate synthetase. Medium 132 (15) was used for P4XB2A42 cultures. Arginine 50 (ug/ml) was added for growth of arginine auxotrophic strains. Spheroplast formation. Spheroplasts were prepared by adapting the method of Dueli et al. (12). Exponentially growing cells were harvested (after addition of cycloheximide, 2 ,ug/ml) at a maximal absorbance of 0.60 at 660 nm measured with a Beckman B spectrophotometer, which represents about 5 x 106 cells per ml. The cells were washed three times with 'stilled water and treated as described by Kovac et al. (20) with 5 volumes (vol/wt of wet cells) of 0.5 M mercaptoethanol-0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 9.3) during 5 min at 300C on a rotary shaker. Cells were washed twice with distilled water and then with a solution (incubation solution) containing 1.8 M sorbitol-10 mM imidazole-HCl (pH 6.4). The cells were suspended in 3 volumes (vol/wt) of the latter solution, and 0.4 ml of Helicase (Industrie Biologique Francaise, Gennevilliers, France) was added per g of wet cells. Conversion to apheroplasts was performed at 30°C with mild shaking and was foUowed by counting the cels diluted (1:400) in the lysis solution containing 0.3 M sorbitol, 0.3 M mannitol, 0.1% bovine serum albumin, 1 mM ethylenediaminetetraacetate (EDTA), disodium salt, 0.1% Tween 80, and 50 mM Tris-hydrochloride (pH 7.5). Conversion was complete in about 90 min. The counting of cells diluted in the incubation solution showed that they did not burst during Helicase treatment. The spheroplasts were pelleted by centrifugation at 4,500 x g at 0 to 4°C for 5 min and were washed twice with a solution containing 1.8 M sorbitol, 0.1% bovine serum albumin, 1 mM EDTA, disodium salt, and 10 mM imidazole-HCl (pH 6.4). The spheroplast pellet was used immediately in cell fractionation or stepwise homogenization experiments.

Spheroplast disruption and fractionation (Fig. 2). The washed spheroplasts were disrupted at 0 to 4°C by suspending them in 7 to 8 volumes (vol/wt) of the above-stated lysis solution and by manual shaking with a glass rod for 1 min and then with a Vortex apparatus (Scientific Industries Inc., model K-550GE), speed 6, for 15 s. The suspension was brought to 20°C and gently homogenized in a Potter homogenizer for 3 min. The rod was driven by a motor (type RM17, Janke and Kunkel KG, Staufen i. Breisgau, Germany) at a speed corresponding to power I, 120. A sample was taken to determine enzymatic activities in the "spheroplast lysate." Whole cells and debris were pelleted by two centrifugations at 750 x g for 10 min at room temperature. Subsequent steps were carried out at 0 to 4°C. The pellet, referred to as "750 x g pellet," was washed by suspension in a solution containing 1 M sorbitol-10 mM imidazole-HCl, pH 6.4, and was recovered by centrifuging at 30,000 x g (average) for 10 min. The 750 x g supernatant, denoted "decanted lysate," was centrifuged at 30,000 x g (average) for 10 min at 0 to 40C to separate fractions referred to as "supernatant" and "particulate pellet." Particulates were rinsed without suspension with a solution containing 0.5 M sorbitol, 0.5 M mannitol, 0.1% bovine serum albumin, 1 mM EDTA, disodium salt, and 50 mM Tris-hydrochloride (pH 7.5). Both 750 x g and 30,000 x g pellets were dispersed in appropriate amounts of the described lysis solution without Tween 80. All the fractions were twice subjected to French press treatment prior to the enzymatic tests. Stepwise homogenization of spheroplast lysate. The method used to homogenize the spheroplast lysate was adapted from Ryan et al. (35). Spheroplasts suspended in 5 volumes (vol/wt) of the described lysis solution were disrupted by an osmotic shock, but with 0.15 M sorbitol and 0.15 M mannitol in the lysis solution for osmotic protection. Homogenization treatment was then applied with an Ultra-Turrax apparatus (type TP18-10, Janke and Kunkel KG, IKA Werk, Staufen i. Breisgau, Germany) (see legend of Fig. 4). Samples were removed after osmotic shock and after defined periods of homogenization, and osmotic protection was brought to 0.6 M by adding an identical CELLS



FIG. 2. Scheme of the fractionation procedure.

VOL. 133, 1978


volume of lysis solution containing 0.45 M sorbitol and 0.45 M mannitol. A sample of the osmotic shock lysate was passed through a French press (Aminco, Silver Spring, Md., 40 kg/cm2). All samples were then centrifuged at 30,000 x g (average) for 20 min. The resulting supernatants were used as enzyme sources, and enzyme activities were expressed as a percentage of the activities obtained in the French press lysate supernatant. Isopycnic gradient analysis. A particulate pellet was obtained exactly as described above and was gently suspended with a Potter homogenizer in a min-

imal volume of a solution containing 20% sorbitol (wt/vol), 0.1% bovine serum albumin, 1 mM EDTA, disodium salt, and 50 mM Tris-hydrochloride, pH 7.5. A 2-ml amount of the suspension was subjected to equilibrium centrifugation in a 20 to 75% (wt/vol) linear sorbitol gradient of 24 ml containing 1 mM EDTA, disodium salt, and 50 mM Tris-hydrochloride, pH 7.5. Centrifugation was carried out with an L-5 Beckman ultracentrifuge in an SW25.1 rotor at 21,000 rpm (45,000 x g at the bottom) for 2 h. These conditions were sufficient to reach equilibrium since a 16-h centrifugation gave identical sedimentation patterns. The gradient was manually pierced and divided into fractions of 1.3 ml. Portions of the fractions were removed for determinations of refractive index and cytochrome oxidase activity. The remainder was diluted six times in a solution containing 10 mM MgC12 and 50 mM Tris-hydrochloride (pH 7.5) and was passed twice through a French pressure cell. Enzyme assays. Enzyme sources were used right away for measurements of ornithine carbamoyltransferase and arginase activities. Portions were desalted on Sephadex G-25 against 20 mM Tris-hydrochloride buffer (pH 7.5)-10 mM MgCl2 for acetylglutamate

synthase, acetylglutamate kinase, acetylglutamylphosphate reductase, acetylornithine aminotransferase, acetylornithine glutamate acetyltransferase, and carbamoylphosphate synthetase assay, against 20 mM triethanolamine-HCl buffer (pH 7.5) for argininosuccinate synthetase and argininosuccinate lyase assay, and against 10 mM imidazole-HCl buffer (pH 7.2) for ornithine aminotransferase assay. Cytochrome oxidase assays were performed in portions not passed through the French press and not desalted. Glucose-6phosphate dehydrogenase and citrate synthase activities were measured on nondesalted and all desalted fractions.

Acetylglutamate synthase was assayed as described by Haas et al. (16), including purification of L-[U-'4C] glutamic acid, with the following modifications in assay mixture: 200 mM Tris-hydrochloride buffer (pH 9.0) and 20 mM [14C]glutamic acid (specific activity, 300 to 400 cpm/nmol). Incubations were carried out at 30°C for 10 min. Since the enzyme is completely inhibited by an excess of L-arginine (52), standard incubation mixtures containing 5 mM L-arginine were used for blank values. Radioactivity was measured in a liquid scintillation spectrometer (Beckman model LS-100) with an efficiency of 55%. Acetylglutamate kinase yielded acetylglutamylphosphate which was converted as it was formed to acetylglutamyl hydroxamate in the presence of hydroxylamine. The hydroxamate was determined by


the ferric chloride method (23). The incubation mixture (prepared at 0°C) consisted of 50 mM Tris-hydrochloride (pH 7.5), 400 mM NH20H HCI (freshly neutralized), 80 mM neutralized acetylglutamate, 10 mM MgCl2, 20 mM ATP (disodium salt, neutralized), and desalted extract, in a final volume of 1 ml. The reaction was started by transfer to 30°C and was stopped after 90 min by addition of a solution containing 10% (wt/vol) FeCl3 6H20, 6% (wt/vol) trichloroacetic acid, and 0.3 M HCI. The precipitate was removed by centrifugation. The absorbance of the hydroxamateFe3+ complex was measured at 510 nm. Under these conditions 1 ,umol of N-glutamyl-5-hydroxamate gave an absorbance of 0.222 (1-cm light path). Since an excess of L-arginine inhibits the enzyme activity almost completely (9, 17), standard incubation mixtures containing 5 mM L-arginine were used for blank values (see Results). The enzyme activity was a linear function of both incubation time (up to about 4 h) and protein concentration, provided that not more than approximately 2 Amol of hydroxamate was fonned. The assay of acetylglutamyl-phosphate reductase was based on the spectrophotometric measurement of the reduction of nicotinamide adenine dinucleotide phosphate (NADP) at 340 nm (47). The reaction mixture (1 ml) consisted of 175 mM Tris-hydrochloride buffer (pH 8.5), 20 mM neutralized N-acetyl-L-glutamyl-y-semialdehyde, 2 mM NADP, 25 mM neutralized potassium arsenate, 10 mM MgCl2, and desalted extract. The presence of MgCl2 in desalting buffer was essential to retain activity. For the assay of acetylornithine aminotransferase, a portion of the desalted extract was diluted to 0.35 ml with 0.1 M potassium phosphate buffer (pH 8.0) to which was added 0.05 ml of pyridoxal phosphate (4 mM), 0.05 ml of potassium a-ketoglutarate (0.2 M), and 0.05 ml of N-acetyl-L-ornithine (0.2 M). All additions were carried out at 00C, and incubation was started by transfer to 30°C and maintained during 30 min. Then the procedure described by Vogel and Jones (46) was closely followed. L-Ornithine acetyltransferase was assayed in the same way as N-acetylglutamate synthase, except that the buffer was 100 mM Tris-hydrochloride (pH 7.5) and acetyl CoA was replaced by 20 mM N2-acetyl-Lornithine. Incubations were carried out for 60 min. In blanks, the acetyl donor was omitted. Carbamoylphosphate synthetase belonging to the arginine pathway was assayed according to Thuriaux et al. (43), but 5 mM UTP was included in the reaction mixture to inhibit the remaining activity of the pyrimidine pathway-specific carbamoylphosphate synthetase. Ornithine carbamoyltransferase was assayed according to Messenguy et al. (25). Inhibition of ornithine carbamoyltransferase by arginase does not occur under our conditions because arginase is not induced and arginine is absent. Activity of argininosuccinate synthetase was determined by coupling the synthesis of argininosuccinate with its subseqNent transformation to arginine by the argininosuccinate lyase of the extract (C. Hennaut, personal communication). To be sure argininosuccinate lyase was not limiting, an excess of coupling enzyme, prepared from strain 100c7, was added in the assay. Reaction mixture (1 ml) contained 0.35 mM



triethanolamine-HCl buffer (pH 7.5), 10 mM MgCl2, 5 mM neutralized ATP, 10 mM neutralized aspartic acid, and desalted enzyme preparation. All additions were made at 0'C; reactions were started by transfer at 30°C. After 60 min of incubation, reactions were stopped by adding 1 mM trichloroacetic acid (10%) to each tube. The precipitated proteins were removed by centrifugation, and the amount of arginine formed was determined on a sample of the supernatant. The argininosuccinate lyase reaction mixture (1 ml) contained 25 mM triethanolamine-HCI buffer (pH 7.5), 10 mM neutralized argininosuccinic acid, and desalted enzyme source. Reactions were started by transfer to 30°C. Incubations were carried out for 30 min and were stopped by adding 1 ml of trichloroacetic acid (10%) to each tube. Proteins were removed by centrifugation. The amount of arginine formed was determined on a sample of the supernatant by the method of Sakaguchi (32). Ornithine transaminase activity was measured as described by De Hauwer et al. (10), with slight modifications. The reaction mixture (3 ml) contained 0.07 mM pyridoxal phosphate, 33 mM imidazole buffer (pH 7.2), 33 mM ,-ornithine, 7 mM a-ketoglutarate, and desalted enzyme preparation. Reactions were stopped after 30 to 60 min of incubation at 30°C, by addition of 1 ml of trichloroacetic acid (10%). The precipitate was removed by centrifugation. A 0.2-ml amount of oaminobenzaldehyde solution (see below) was added to 2.8 ml of supernatant. The color was developed at 30'C for 1 h, and absorbance was measured at 430 nm. A blank with trichloroacetic acid and extract was necessary when activity was low. The solution of oaminobenzaldehyde was freshly prepared by dissolving 50 mg in 0.5 ml of ethanol, and it was brought to 10 ml with water. Arginase was assayed as described by Messenguy et al. (25) and citrate synthase (EC, as described by Parvin (28). Glucose-6-phosphate dehydrogenase (EC was assayed by the method of Domagk (personal communication) by recording the NADP reduction at 340 nm. One milliliter of reaction mixture contained 10 mM MgCl2, 0.4 mM NADP, 1 mM glucose-6-phosphate, 1 mM Mg-EDTA, 0.5 mM mercaptoethanol, and 0.1 M triethanolamine-HCI (pH 7.5). The reaction was started by adding the enzyme. Cytochrome oxidase (EC was assayed as described by Smith (41) with 20 mM ferrocytochrome c and with 50 mM Tris-hydrochloride-1 mM EDTA (disodium salt), pH 7.4, as buffer assay (38). Protein was estimated by the method of Lowry et al. (24), with bovine serum albumin as the standard. Chemicals. ["'C]NaHCO:l and L_[U-_4C]glutamic from the Radiochemical Centre, Amersham, England.

L-Glutamate, N2-acetyl-L-ornithine, L-ornithine hydrochloride, L-arginine hydrochloride, and amino oxyacetic acid ½2HCl were from Sigma Chemical Co., St. Louis, Mo. N-acetyl-L-glutamyl-y-semialdehyde was enzymatically synthesized by the method of Vogel and MacLellan (47) except that dialysis was performed on a Sephadex G-25 column. E. coli strain P4XB2A42 has served as source of acetylornithine aminotransferase. o-Aminobenzaldehyde was prepared as described by Smith and Opie (42). N-acetyl-L-glutamate was from Sigma or from Aldrich Chemical Co., Mil-


waukee, Wis. Dowex 50-W (X 8, 200 to 400 mesh, H+ form) was purchased from Fluka, Buchs, Switzerland. Sephadex G-25 was a product of Pharmacia, Uppsala, Sweden. D-Sorbitol was from Merck, Darmstadt, Germany, and D-mannitol was from Difco Laboratories, Detroit, Mich. Cytochrome c and acetyl CoA were products of Boehringer, Mannheim, Germany. Cytochrome c was reduced with dithionite and twice desalted to remove excess dithionite. Tween 80 was a product of Atlas-Goldschmidt, Essen, Germany. Helicase was from Industrie Biologique Fran9aise, Gennevilliers, France. Nucleotides, carbamoylphosphate, and most other chemicals were from Sigma, Boehringer, or Merck.

RESULTS Subcellular distribution of enzymes. In the fractioning experiments of Tables 1 and 2, cytochrome oxidase, citrate synthase, and glucose-6-phosphate dehydrogenase were taken as markers of the mitochondrial inner membrane, of the mitochondrial matrix (1, 27, 35), and of the cytosol (18, 31, 35), respectively. From the distribution of glucose-6-phosphate dehydrogenase and citrate synthase in the second and third columns of Table 1, both the lysis efficiency and the portion of the initial mitochondrial material present in the decanted lysate can be seen. The last column represents the particulate fraction, where all the cytochrome oxidase activity present in the decanted lysate and 70 to 80% of the citrate synthase activity were found. In contrast, glucose-6-phosphate dehydrogenase resulting from the decanted lysate was quantitatively recovered in the supernatant as well as the solubilized portion of citrate synthase. The specific activities of citrate synthase and cytochrome oxidase were increased significantly in the particulate pellet (Table 2). A major part of the initial mitochondrial material was thus found in the particulates. In contrast, in preliminary experiments it was observed that in spite of almost 100% efficiency of spheroplast lysis the recovery of mitochondrial enzymes in the particulate pellet was sometimes very low and, above all, not reproducible. Others have also come up against this problem (13, 21, 31). Taking citrate synthase as a reference, we observed that, when the lysate was twice decanted by centrifugation at low speed to eliminate whole cells, spheroplasts, debris, and nuclei, the mitochondria also pelleted. The loss could reach 90%, whereas no more than 10% appeared in the particulates (not shown). The improvement of mitochondria recovery was studied while the constraints of a high osmotic protection (0.3 M sorbitol plus 0.3 M mannitol) and an absence of strong mechanical shaking were maintained during the lysis process. The best results were obtained by the combined ef-


VOL. 133, 1978


TABLE 1. Subcellular distribution of enzymes as a percentage of activity (data in Tables I and 2 derive from the same experiments)


750 x 9








Cytochrome oxidase

Glucose-6-phosphate dehydrogenase Acetylglutamate synthase

Arginine metabolism in Saccharomyces cerevisiae: subcellular localization of the enzymes.

Vol. 133, No. 3 JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1096-1107 0021-9193/78/0133-1096$02.00/0 Copyright ) 1978 American Society for Microbiology P...
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